Nephrin gene and protein

ABSTRACT

The present invention provides for compositions and methods for detecting susceptibility for basement membrane disease, in particular congenital nephrotic syndromes of the Finnish type. The present invention provides for nucleic acids and protein for use in methods and compositions for the diagnosis of disease and identification of small molecule therapeutics for treatment of such disease, in particular of proteinuria associated with kidney disease.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.09/759,622, filed Jan. 12, 2001, which is a divisional of U.S.application Ser. No. 09/040,774, filed Mar. 18, 1998 now U.S. Pat. No.6,207,811, issued Mar. 21, 2001. The entire teachings of the aboveapplications are incorporated herein by reference.

BACKGROUND OF THE INVENTION

Congenital nephrotic syndrome of the Finnish type (CNF, NPHS1, MIM256300) is an autosomal recessive disorder, and a distinct entity amongcongenital nephrotic syndromes. It is characterized by massiveproteinuria at the fetal stage and nephrosis at birth. Importantly,NPHS1 appears to solely affect the kidney and, therefore, it provides aunique model for studies on the glomerular filtration barrier.

The primary barrier for ultrafiltration of plasma in renal glomerulicomprises three layers; a fenestrated endothelium, a 300-350 nm thickglomerular basement membrane (GBM), and slit pores, i.e. diaphragmslocated between the foot processes of the epithelial cells. This barrieris a highly sophisticated size-selective molecular sieve whose molecularmechanisms of function are still largely unclarified. It is anticipatedthat the GBM, a tightly cross-linked meshwork of type IV collagen,laminin, nidogen and proteoglycans, contains pores that restrict thepenetration of large proteins and cells, and, additionally, it has beenhypothesized that anionic heparan sulfate proteoglycan componentscontribute to an electric barrier for macromolecules (Kasinath andKanwar, 1993). The glomerular filter is affected in a large number ofacquired and inherited diseases resulting in extensive leakage of plasmaalbumin and larger proteins leading to nephrotic syndrome and end stagerenal disease. Understanding of the molecular mechanisms of theglomerular filtration process and its pathology is of fundamentalimportance for clinical medicine, which, in turn, may facilitate noveldevelopments for diagnosis and treatment of complications in primary andsecondary diseases of the kidney. Genetic diseases with defects in thefiltration barrier as major symptoms can serve as models for providingsuch knowledge.

Congenital nephrotic syndromes (NPHS) form a heterogenous group ofdiseases characterized by massive proteinuria at or shortly after birth(Rapola et al., 1992). Nephrotic syndrome can be primary, acquired, or apart of other syndromes. Congenital nephrotic syndrome of the Finnishtype (CNF, NPHS1) is a distinct entity among NPHS. It is an autosomalrecessive disorder with an incidence of 1:10,000 births in Finland, butconsiderably less in other countries (Norio, 1966; Huttunen, 1976). Thedisease manifests itself already at the fetal stage with heavyproteinuria in utero, demonstrating early lesions of the glomerularfiltration barrier. The pathogenesis of NPHS1 has remained obscure.There are no pathognomonic pathologic features, the most typicalhistological finding of NPHS1 kidneys being dilation of the proximaltubuli (Huttunen et al. 1980). The kidneys are also large and have beenfound to contain a higher amount of nephrons than age-matched controls(Tryggvason and Kouvalainen, 1975). Electron microscopy reveals noabnormal features of the GBM itself, although there is a loss of footprocesses of the glomerular epithelial cells, a finding characteristicfor nephrotic syndromes of any cause. Analyses of GBM proteins, such astype IV collagen, laminin, and heparan sulfate proteoglycan have notrevealed abnormal findings in NPHS1 (e.g. see Ljungberg et al. 1993,Kestilä et al. 1994a). NPHS1 is a progressive disease, usually leadingto death during the first two years of life, the only life-savingtreatment being kidney transplantation (Holmberg et al. 1995).Importantly, most transplanted patients have, thus far, not developedextrarenal complications, suggesting that the mutated gene product ishighly specific for kidney development and/or glomerular filtrationfunction. However, about 20% of the patients have developedpost-transplantation nephrosis the cause of which is unknown (Laine etal., 1993; Holmberg et al., 1995).

Due to its high specificity for the glomerular filtration process, NPHS1provides a unique model disease for studies on this important kidneyfunction. Since there was no strong candidate gene for the disease, wehave used the positional cloning approach in our attempts to identifythe CNF gene, and have localised the gene to a 150 kb region onchromosome 19q13.1 (Kestilä et al., 1994b; Männikkö et al., 1995). Wehave identified a novel gene in the critical region and shown it to bemutated in NPHS1. The gene product is a novel transmembrane protein,which in the human embryo shows a high expression level in renalglomeruli.

SUMMARY OF THE INVENTION

The present invention provides for the novel protein Nephrin and thegene encoding for this protein. The present invention encompasses anovel DNA nucleic acid sequence which is the nucleic acid sequence ofSEQ ID NO: 1 which encodes for the nephrin protein. The presentinvention also encompasses the protein encoded for by the coding regionsof the nucleic acid sequence of SEQ ID NO: 1 which has the amino acidsequence of SEQ ID NO: 2. In particular, the present invention alsoencompasses the mature nephrin protein in which the signal peptide hasbeen cleaved off.

The present invention encompasses method, reagents and kits forscreening individuals for the presence of mutated Nephrin gene fordiagnosis, pre-natal screening, or post-natal screening forsusceptibility to glomerular nephrosis or basement membrane disease. Inparticular, the present invention provides for screening for congenitalnephrotic syndromes of the Finnish type (NPHS1).

The present invention provides for methods, reagents and kits for thetherapeutic treatment of basement membrane disease associated withdefective endogenous Nephrin gene product. Thus the present inventionprovides for therapeutic treatment using Nephrin protein, and inparticular using protein produced by recombinant DNA methods. Inaddition, the present invention provides for gene therapy usingtherapeutic nucleic acid constructs containing the Nephrin gene, orsubstantially similar DNA sequence thereto.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will be better understood in view of the attached drawingswherein:

FIG. 1 is a drawing showing a physical map of the NPHS1 locus at 19q13.1and genomic organization of the NPHS1 gene. FIG. 1A, is a physical mapof the 920 kb region between markers D19S208 and D19S224. FIG. 1B, is adiagram of overlapping cosmid clones spanning the 150 kb critical regioncontaining the NPHS1 gene. Location of polymorphic markers are indicatedby arrows. FIG. 1C, is a diagram showing the location of five genes,NPHS1, APLP1, A, B, C, characterised and searched for mutations in thisstudy. FIG. 1D, is a drawing showing a schematic structure of the NPHS1gene.

FIG. 2 shows a northern blot analysis of nephrin expression (the NPHS1gene product) with mRNA from human embryonic and adult tissues. Thenorthern filters containing 2 ug of human poly(A) RNA from four fetaland eight adult tissues (Clontech) were hybridized with a 1,371 bpnephrin cDNA probe (exons 1-10) made by RT-PCR from fetal kidney poly(A)RNA. FIG. 2A, shows distinct expression can be seen only with fetalkidney RNA (arrow). FIG. 2B, shows results using RNA from adult tissues,intense signal is only observed in a 4.3 kb band with kidney RNA(arrow), the other tissues exhibiting only insignificant if any positivesignals. The tissues studied are marked above the filter and molecularsize markers (kb) are shown to the sides of the filters.

FIG. 3 is a diagram of mutation analysis of the NPHS1 gene. Left: (A)Pedigree of an NPHS1 family with an affected child having a 2-bpdeletion in exon 2. Sequences of the deletion point shown from patient(homozygous), parent (heterozygous) and a healthy sibling. Right: (B)Pedigree of an NPHS1 family with an affected child having a nonsensemutation in exon 26. Sequences of the mutated region are shown frompatient (homozygous), parent (heterozygous) and a healthy sibling.

FIG. 4 is a diagram of the nucleotide-derived amino acid sequence ofnephrin (the NPHS1 gene product) and predicted domain structure. FIG. 4A(SEQ ID NO: 2), is the predicted N-terminal signal sequence is 22residues, the cleavage site being marked with an arrow. A putativetransmembrane domain (spanning residues 1,059-1086) is shown in bold andunderlined. The putative extracellular part of the protein containseight Ig-like modules (boxed), and one fibronectin type III-like moduleadjacent to the transmembrane domain (boxed with a bold line, residues941-1025). Cysteine residues are indicated by black dots and the tenputative N-glycosylation sites in the extracellular part of the proteinare underlined. FIG. 4B shows the predicted domain structure of normalnephrin and the predicted effects of the two mutations (Fin-major andFin-minor) identified in this study. The Ig-like modules are depicted bypartial circles and the fibronectin type III like-motif by a hexagon.The transmembrane domain is shown as a black rectangle located in amembrane lipid bilayer. The locations of two free cysteine residues areindicated by lines with a black dot at the end. The Fin-major mutationwould result in the production of part of the signal peptide and a shortnonsense sequence. The Fin-minor mutation would result in a nephrinmolecule lacking a part of the cytosolic domain.

FIG. 5 shows the results of expression of nephrin mRNA in humanembryonic kidney by in situ hybridization. FIG. 5A, shows intenseexpression in glomeruli throughout the renal cortex, little if anyspecific expression being observed in other structures. (4× objectivemagnification). FIG. 5B, is a view at higher magnification which revealsintense expression in the periphery of individual glomeruli (straightarrows), probably mainly in epithelial cells. No expression is observedin the Bowman's capsule (bent arrow), proximal tubuli (open arrows), orendothelial cells of vessel walls. (20× objective magnification).

DETAILED DESCRIPTION OF THE INVENTION

Congenital nephrotic syndrome of the Finnish type (CNF, NPHS1, MIM256300) is an autosomal recessive disorder, and a distinct entity amongcongenital nephrotic syndromes. It is characterized by massiveproteinuria at the fetal stage and nephrosis at birth. Importantly,NPHS1 appears to solely affect the kidney and, therefore, it provides aunique model for studies on the glomerular filtration barrier. The NPHS1gene has been localized to 19q13.1, and in the present study linkagedisequilibrium was used to narrow the critical region to 150 kilobaseswhich were sequenced. At least 10 novel genes, and one encoding amyloidprecursor like protein were identified in this region. Five of thegenes, all of which showed some expression in kidney, were analyzed bysequencing all their 63 exons in NPHS1 patients. Two mutations, a 2-bpdeletion in exon 2 and a single base change in exon 26, both leading topremature stop codons were found in a novel 29-exon gene. The mutationswere found either as homozygous or compound heterozygous in 44 out of 49patients, 4 patients having the 2 bp deletion in one allele, the otherpotential mutation still being unknown. None among controls was foundhomozygous or compound heterozygous for the mutations. The gene product,termed nephrin, is a 1,241-residue putative transmembrane protein of theimmunoglobulin family of cell adhesion molecules which by northern andin situ hybridization was shown to be kidney glomerulus-specific. Theresults demonstrate a crucial role for nephrin in the development orfunction of the kidney filtration barrier.

The invention will be more clearly understood by examination of thefollowing examples, which are meant by way of illustration and notlimitation.

EXAMPLE 1

Methods and Procedures

Sequencing of Cosmid Clones

Isolation of cosmid clones spanning the region between D19S208 andD19S608 has been reported previously (Olsen et al., 1996). DNA of cosmidclones F19541, R33502, F15549, R28051, F19399, R31158 and R31874 wasmechanically sheared by nebulization and fragments of 1000-2000 bp wereisolated and subcloned into M13 phage, prior to random sequencing usingABI 377 automated DNA sequencers.

Analysis of Sequence

In order to develop new microsatellite markers, repeat regions weresearched from the sequence, and three of them (D19S1173, D19S1175,D19S1176) were found to be polymorphic. Homology comparisons wereperformed using BLASTX and BLASTN programs (Altschul et al., 1990).Prior to BLASTN analyses, the nucleotide sequence was filtered usingCENSOR (Jurka et al., 1996) to mask out repeat regions like Alusequences. Exon prediction was made using GRAIL II (Uberbacher andMural, 1991), GENSCAN (Burge and Karlin, 1997), FGENEH and HEXON(Solovyeh et al., 1994) programs, and prediction of the proteinstructure was made using BLASTP (Altschul et al., 1990) and EXPASYmolecular biology server (Appel et al., 1994). The mutation search wasperformed by comparing patient sequences to the normal genomic sequenceusing the FASTA program of the GCG package (Genetics Computer Group,1996).

Isolation of cDNAs

cDNAs were generated by PCR from poly(A) RNA from different tissuesusing primers based on the exon sequences. The PCR fragments weresequenced and used for screening of cDNA libraries. Marathon ready cDNAkits (Clontech Laboratories) were also used to characterize the 5′ and3′ extremities of the cDNAs. Comparison of the cDNA and genomicsequences were made to establish the sizes of introns, as were intronsequences at acceptor and donor splice sites.

Southern and Northern Blots and in Situ Hybridization Analyses

For Southern analyses samples containing 10 μg of genomic DNA weredigested with different restriction enzymes and electrophoreses on 1%agarose gels, transferred to nylon membranes and hybridized with thecDNA probe. In multiple-tissue northern analysis poly(A) RNAs from 8adult and 4 fetal tissues were studied (Clontech). Hybridization wasdone in ExpressHyb buffer at 65° C. using a cDNA clone containing exons1-10.

For in situ hybridization a fragment from the NPHS1 cDNA clone(corresponding to exon 10) was labeled with digoxigenin (BoehringerMannheim), cut to about 150 base pair fragments by alkaline hydrolysis,and then used as a probe. Tissue sections of 7 μm from a 23-week humanembryonic kidney were treated with 0.2M HCl, 0.1M triethanolaminebuffer, pH 8.0, containing 0.25% (v/v) acetic anhydride and 100 μg/mlproteinase K. The sections were hybridized with the probe at 62° C. for16 h. After rinsing in 50% formamide and standard sodium citrate, theprobe was immunologically detected with an antibody to digoxigeninconjugated to alkaline phosphate enzyme (Boehringer Mannheim). The colorwas developed with NBT and BCIP.

Mutation Analysis

In this study we analyzed 49 Finnish NPHS1 patients, their parents and atotal of 54 healthy siblings. The diagnosis of NPHS1 is based on severeproteinuria, a large placenta (>25% of birth weight), nephrotic syndromeduring the first weeks of life, and exclusion of other types ofcongenital nephrotic syndrome (Koskimies 1990). Additionally, samplesfrom 83 control individuals were analysed.

The NPHS1 gene was analysed by PCR-amplifying and sequencing all exonregions from genomic DNA. The sequences of the primers for exon 2 were5′GAGAAAGCCAGACAGACGCAG3′ (5′ UTR)(SEQ ID NO: 3) and5′AGCTTCCGCTGGTGGCT3′ (intron 2) (SEQ ID NO: 4), and the sequences ofthe primers for exon 26 were 5′CTCGGGGAGACCCACCC3′ (intron 23) (SEQ IDNO: 5) and 5′CCTGATGCTAACGGCAGGGC3′ (intron 26) (SEQ ID NO: 6). PCRreactions were performed in a total volume of 25 ul, containing 20 ng oftemplate DNA, 1× AmpliTaq buffer (Perkin-Elmer), 0.2 mM of eachnucleotide, 50 ng of primers and 0.5 U AmpliTaq Gold DNA polymerase. Thereactions were carried out for 30 cycles with denaturation at 95° C. for1 min, annealing at 60° C. for 1 min, and extension at 72° C. for 1 min.In the first cycle denaturation was carried out for 12 min, andextension in the last cycle was for 8 min. PCR products were separatedby 1.5% agarose gel, sliced off and purified by the QiaexII system(Qiagen). The purified PCR product was sequenced using specific primersemploying dRhodamine dye-terminator chemistry and an ABI377 automatedsequencer (Perkin-Elmer).

When screening for the NPHS1 Fin-major mutation from parents, siblingsand controls, a 100 bp PCR product containing the exon 2 deletion sitewas amplified using a radioactively end-labeled primer, andelectrophoresed on 6% polyacrylamide gels. The second NPHS1 Fin-minormutation could be screened for using a novel restriction site for DdeI.The 140 bp amplified PCR product was digested with DdeI and the products(140 bp or 90 bp+50 bp) were separated on an agarose gel (1% SeaKemagarose-3% NuSieve agarose).

In general, methods and procedures for performing molecular biologicaland biochemical techniques are known in the art and can be found inavailable texts and references, such as for example Sambrook et al.,(1989) Molecular Cloning: a laboratory manual, 2nd edition (Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y.); Short Protocols inMolecular Biology, 2nd edition (edited by Ausubel et al., John Wiley &Sons, New York, 1992); Davis et al., (1986) Basic Methods in MolecularBiology (Elsevier, N.Y.); Gene Expression Technology (edited by DavidGoeddel, Academic Press, San Diego, Calif., 1991).

EXAMPLE 2

Characterization of Genes at the CNF Locus

Following localisation of the NPHS1 gene to 19q13.1, overlapping cosmidclones from the interval of interest between markers D19S208 and D19S224were isolated (Männikkö et al. 1995; Olsen et al., 1996). Based on thesignificant linkage disequilibrium observed with D19S608 and D19S610, aswell as the new microsatellite markers, D19S1173, D19S1175, andD19S1176, identified in this study, the NPHS1 gene was fine-mappedbetween D19S1175 and D19S608, in close vicinity of D19S1176 and D19S610(FIG. 1). Southern hybridization analyses of NPHS1 patient DNA withgenomic clones did not reveal variations, suggesting that the NPHS1mutations do not represent major genomic rearrangements. The 150 kbcritical region was sequenced in its entirety, and the sequence wassearched for potential candidate genes using exon prediction programsand data base similarity searches. Based on those analyses, the criticalregion was estimated to include over 100 potential exons. Similaritysearches revealed one previously known gene, i.e. APLP1 encoding anamyloid precursor-like protein (Lenkkeri et al., in press) and eightdistinct expressed sequence tags (ESTs). Together the analyses indicatedthe presence of at least ten novel genes in the critical region.

FIG. 1 illustrates a physical map of the NPHS1 locus at 19q13.1 andgenomic organisation of the NPHS1 gene. FIG. 1A, Physical map of the 920kb region between D19S208 and D19S224. FIG. 1B, Overlapping cosmidclones spanning the 150 kb critical region containing the NPHS1 gene.Location of polymorphic markers are indicated by arrows. FIG. 1C,Location of five genes, NPHS1, APLP1, A, B, C, characterised andsearched for mutations in this study. FIG. 1D, Schematic structure ofthe NPHS1 gene.

Using Grail and Genscan exon prediction programs and sequences fromcDNAs, the exon/intron structures of five of the genes, NPHS1 (FIG. 1),APLP1, A, B, and C (not shown) were determined. Although steady statetranscript levels varied, northern analyses revealed expression of allthe genes in kidney, and with the exception of NPHS1, also in othertissues. Therefore, none of them could be excluded as the NPHS1 gene andall were subjected to mutation analysis.

EXAMPLE 3

Identification of the NPHS1 Gene

Haplotype analyses of NPHS1 chromosomes have revealed two major classesin Finnish patients (Männikkö et al., 1995; this study). The first onecontaining haplotypes 1-1-1-6-g-2-8-9 and 1-1-1-6-g-6-4-2 (markersD19S1173, D19S1175, D19S1176, D19S610, RFLP of gene B, D19S608, D19S224,D19S220, respectively) is the most common one found in 78% of FinnishNPHS1 chromosomes. The second haplotype class, 3-5-3-6-a-8-10-x, isfound in 13% of cases. The remaining 9% of observed haplotypes showtotally different allele combinations, and have been thought torepresent other mutations. Two major haplotype classes could representthe same mutation, because they both share allele 6 of D19S610. However,the present results demonstrated that they represent two differentmutations.

Since Southern hybridization analyses did not reveal any major generearrangements, mutations were searched by direct sequencing ofPCR-amplified exon regions of, if necessary, all the genes of thisregion.

The 17 exon APLP1 gene located distal to D19S610 did not show variationsbetween patients and controls, and was excluded as the NPHS1 gene(Lenkkeri et al., in press). Also, the novel genes A, B and C,containing 9, 5 and 3 exons, respectively, did not have sequencevariants segregating with NPHS1, and could similarly be excluded as theNPHS1 genes (data not shown). A fourth novel gene (NPHS1) locatedproximal to D19S610 encoding a transcript of about 4.3 kb was shown tobe strongly expressed in human embryonic and adult kidneys, no clearsignals above background being observed in other tissues (FIG. 2).

FIG. 2 illustrates the results of Northern analysis of nephrinexpression with mRNA from human embryonic and adult tissues. Thenorthern filters containing 2 ug of human poly(A) RNA from four fetaland eight adult tissues (Clontech) were hybridized with a 1,371 bpnephrin cDNA probe (exons 1-10) made by RT-PCR from fetal kidney poly(A)RNA. In FIG. 2A, Distinct expression can be seen only with fetal kidneyRNA (arrow). In FIG. 2B, Using RNA from adult tissues, intense signal isonly observed in a 4.3 kb band with kidney RNA (arrow), the othertissues exhibiting only insignificant if any positive signals. Thetissues studied are marked above the filter and molecular size markers(kb) are shown to the sides of the filters.

Therefore, this gene was a strong candidate for NPHS1. Full-length cDNAfor the transcript was constructed using fetal kidney poly(A) mRNA(Clontech) and PCR primers made based on the predicted exon structure.The gene was found to have a size of 26 kb and to contain 29 exons (FIG.1).

Exon sequencing analyses revealed the presence of two major mutations inover 90% of NPHS1 chromosomes (FIG. 3). FIG. 3 illustrates mutationanalysis of the NPHS1 gene. Left: (A) Pedigree of a NPHS1 family with anaffected child having a 2-bp deletion in exon 2. Sequences of thedeletion point shown from patient (homozygous), parent (heterozygous)and a healthy sibling. Right: (B) Pedigree of a NPHS1 family with anaffected child having a nonsense mutation in exon 26. Sequences of themutated region are shown from patient (homozygous), parent(heterozygous) and a healthy sibling.

The first mutation, a 2-bp deletion in exon 2 causes a frameshiftresulting in the generation of a stop codon within the same exon. Thismutation was found in all NPHS1 chromosomes with the haplotype1-1-1-6-g-2-8-9 and 1-1-1-6-g-6-4-2 (total of 76 chromosomes). One outof 83 control individuals was heterozygous for the Fin-major mutation.The second sequence variant found in the NPHS1 gene was a nonsensemutation CGA.fwdarw.TGA in exon 26, present in patients with haplotype3-5-3-6-a-8-10-x (13 chromosomes), and three patients with differenthaplotypes. None of the parents, healthy siblings, or controls (total of230 individuals) were homozygous or compound heterozygous for the twomutations identified here. Since the gene cloned in this study is theone involved in a hereditary nephrotic syndrome, we refer to it as NPHS1gene.

Out of 49 NPHS1 patients studied, 32 were homozygous for the 2-bpdeletion in exon 2 (Fin-major), four were homozygous for the nonsensemutation in exon 26 (Fin-minor), and eight were compound heterozygotes.Four patients had the Fin-major mutation in one allele, the otherpotential mutation still being unknown. One patient had neither one ofthe two mutations.

EXAMPLE 4

Characterization of the NPHS1 Gene Product

The cDNA-predicted amino acid sequence of the NPHS1 protein (nephrin) is1,241 residues (FIG. 4), with a calculated molecular mass of 134,742without posttranslational modifications.

FIG. 4 shows nucleotide-derived amino acid sequence of nephrin andpredicted domain structure (the NPHS1 gene product). FIG. 4A illustratesthe predicted N-terminal signal sequence is 22 residues, the cleavagesite being marked with an arrow. A putative transmembrane domain(residues 1,059-1086) is shown in bold and underlined. The putativeextracellular part of the protein contains eight Ig-like modules(boxed), and one fibronectin type III-like module adjacent to thetransmembrane domain (boxed with a bold line). Cysteine residues areindicated by black dots and the ten putative N-glycosylation sites inthe extracellular part of the protein are underlined. FIG. 4Billustrates predicted domain structure of normal nephrin (the NPHS1 geneproduct) and the predicted effects of the two mutations (Fin-major andFin-minor) identified in this study. The Ig-like modules are depicted bypartial circles and the fibronectin type III like-motif by a hexagon.The transmembrane domain is shown as a black rectangle located in amembrane lipid bilayer. The locations of three free cysteine residuesare indicated by lines with a black dot at the end. The major NPHS1mutation would result in the production of a secreted protein containingonly a part of the first Ig-like module. The Fin-minor mutation wouldresult in a nephrin molecule lacking a part of the cytosolic domain.

Several similarity comparison and protein structure prediction programspredicted that the NPHS1 protein would be a transmembrane protein of theimmunoglobulin superfamily. There is a tentative 22-residue-longN-terminal signal peptide, an extracellular domain containing eightimmunoglobulin-like domains, one fibronectin type III domain-likemodule, followed by a single putative transmembrane domain-likesequence, and a cytosolic C-terminal end. In spite of the presence ofknown structural modules (FIG. 4), the sequence identity withcorresponding domains of proteins in the data base was relatively low.The tentative extracellular portion of the protein contains ten NXS orNXT consensus triplets for N-glycosylation. Furthermore, there are sevenSG doublets, that are potential attachment sites for heparan sulfate.

Northern hybridization analysis carried out with poly(A) mRNA from fourhuman embryonic and eight adult tissues revealed a high steady statelevel of the NPHS1 gene transcript in the kidney, but not notably inother tissues. (FIG. 2). In situ hybridization carried out on a kidneysample from a 23-week-old human embryo revealed intense expressionsignals in the glomeruli (FIG. 5A). At higher magnification (FIG. 5B),the signals could be seen in the periphery of mature and developingglomeruli, while the central mesangial regions are negative. It isapparent that the positive cells are epithelial podocytes. No specificsignals were obtained with the antisense control probe.

FIG. 5 illustrates expression of nephrin mRNA in human embryonic kidneyby in situ hybridization FIG. 5A shows intense expression is seen inglomeruli throughout the renal cortex, little if any specific expressionbeing observed in other structures. (4× objective magnification). FIG.5B, Higher magnification reveals intense expression in the periphery ofindividual glomeruli (straight arrows), probably mainly in epithelialcells. No expression is observed in the Bowman's capsule (bent arrow),proximal tubuli (open arrows), or endothelial cells of vessel walls.(20× objective magnification).

EXAMPLE 5

The NPHS1 Gene and its Gene Product Nephrin

Several lines of evidence obtained in the present study show that wehave positionally cloned the gene affected in congenital nephroticsyndrome of the Finnish type. First, the defective gene is located inthe critical 150 kb region on chromosome 19q13.1 to which the gene hasbeen localized using linkage disequilibrium analyses (Kestilä et al.,1994b; Männikkö et al., 1995; Kestilä et al. manuscript). Second, thetwo mutations identified in the study were shown to be present, eitheras homozygous or compound heterozygous mutations, in 44 out of 49Finnish patients studied. Four of the remaining patients had the majormutation in one allele, the mutation in the other allele being, as yet,unidentified. One patient who did not have either of the two mutations,has a unique haplotype and, therefore, probably carries a differentmutation. Third, individuals homozygous or compound heterozygous for themutations were not found in 230 control DNAs. Additional, indirectevidence was the strong and practically renal glomeruli-specificexpression of the gene, which implies involvement of the gene product inglomerular development or function.

Identification of the NPHS1 Gene

The present identification of the NPHS1 gene demonstrates the power oflinkage disequilibrium analysis and direct DNA sequencing in thepositional cloning of disease genes containing small mutations. Here,linkage disequilibrium mapping (Hstbacka et al., 1994) which when usedwith DNA from individuals of a homogenous population, such as theisolated Finnish population (de la Chapelle, 1993), was utilized tolocalize the NPHS1 gene to a 150 kb genomic segment. In order to findgenes located in this region, the entire segment was first sequenced,and using a combination of exon prediction programs and homologycomparison analyses we could construct remarkably accurate genestructures that were verified from cDNAs. These cDNAs could be isolatedeither with the use of EST clones or by using the predicted exonsequences to construct cDNAs by PCR from mRNA. In this manner we couldquickly identify 11 genes within the 150 kb NPHS1 containing genomicsegment. Since none of the genes was an obvious candidate for NPHS1, andno major gene rearrangements, such as deletions, insertions orinversions, were found in patient DNAs, search for small mutations hadto be initiated, if necessary, in all the 11 genes. Having determinedthe exon and cDNA sequences for the genes, methods such as SSCP andDGGE, which are frequently used for identification of small mutations,were potential alternatives. However, our experience from the search forsmall mutations in Alport syndrome (Barker et al., 1990; Tryggvason,1996) suggests that these methods can frequently yield false negatives.For example, SSCP analyses in quite large patient populations haverevealed only a 35-50% mutation detection rate (Kawai et al., 1996,Knebelmann et al. 1996, Renieri et al., 1996), while our directsequencing of PCR-amplified exon regions has yielded over 80% detection.We therefore decided to use direct sequencing of exon regions to findthe NPHS1 mutations. Although we had to sequence numerous exons ofseveral genes, this relatively soon resulted in the identification oftwo small mutations in one gene. We conclude that sequencing of even alarge candidate gene region and direct sequencing of its genes is anattractive and, above all, reliable method to search for small mutationsin positional cloning, particularly if only few mutations can beexpected to be present.

Genetics of NPHS1

Crucial components in the successful positional cloning of the NPHS1gene were the small isolated population, good clinical records andequal, high quality health care system which made it possible toreliably collect family samples. A typical situation in populationisolates is that close to 100% of cases are caused by the same mutation,and this phenomenon can already be seen in haplotype analysis. Observedchanges in the founder haplotype, caused by historical recombinations,can be used to restrict the critical chromosomal region to a shortgenomic segment. Thus, differences in the major NPHS1 haplotype1-1-1-6-g-2-8-9 enabled substantial narrowing of the interval, leadingto the isolation of the NPHS1 gene. The major NPHS1 mutation causes only78% of cases, in contrast to many other “Finnish diseases” with 95-98%prevalence of major disease alleles (e.g. Ikonen et al., 1991). However,the two main NPHS1 mutations characterized in this study togetherrepresent 94% of Finnish cases.

Congenital nephrotic syndrome of the Finnish type is enriched in theFinnish population, but several cases can be found worldwide.Considerable immigration from Finland to Minnesota has also caused thespread of NPHS1 to the USA (Norio 1966; Mahan et al., 1984). Inaddition, several CNF cases have been diagnosed in different Europeancountries, and linkage studies have supported association of analyzedfamilies to the same chromosome 19 locus (Fuchshuber et al., 1996).

The identification of the NPHS1 gene and disease causing mutations haveimmediate clinical significance, as they have enabled the development ofexact DNA-based diagnosis for NPHS1 and carrier screening. This isparticularly important, as we have recently demonstrated that thescreening method widely used in Finland for NPHS1 based on measurementsof alpha-fetoprotein levels in amniotic fluid can lead to false positiveresults and subsequent abortions of healthy NPHS1 carriers (Männikkö etal., 1997).

Nephrin—a Glomerulus-specific Cell Adhesion Receptor

Due to the high association of expression and pathology with glomeruli,the proximal part of the nephron, we have named the NPHS1 gene productnephrin. The role of nephrin remains unknown, but it is likely to be anadhesion receptor and a signaling protein, as its domain structureresembles that of a large group of cell adhesion receptors belonging tothe immunoglobulin superfamily (Brummendott and Rathjen, 1994).

The Ig-like domains of nephrin are all of type C2 which is particularlyfound in proteins participating in cell-cell or cell-matrixinteractions. Between the sixth and seventh Ig-like domains there is aspacer of about 130 residues containing an unpaired cysteine, and thereis another unpaired cysteine in the fibronectin type III-like domain.Their SH groups could be involved in the formation of cishomo/heterodimers, participate in thioether or thioester bonds withunknown structures, or be buried within the domains, as suggested byBrummendott and Rathjen (1994).

Data base searches revealed that the cytosolic domain that contains ninetyrosine residues of nephrin has no significant homology with otherknown proteins. However, sequence motifs surrounding tyrosines suggestthat tyrosines 1176, 1192 and 1217 could become phosphorylated duringligand binding of nephrin (see, Songyang et al. 1993). In that case,binding sites for the SH2-domains of Src-family kinases, Abl-kinase, andan adaptor protein Nck might be created (tyrosines 1176 and 1192 arefollowed by the motif DEV, and tyrosine 1217 by DQV). The crucial rolefor the intracellular domain of nephrin is emphasized by the fact thatthe Fin-minor mutation which results in the loss of 132 out of 155residues results in full blown NPHS1.

The pathogenesis of NPHS1 has been thought to primarily or secondarilyinvolve the highly anionic glycosaminoglycans, as the content of suchmolecules that are considered important for the glomerular filtrationprocess is reported to be decreased in the GBM in proteinuria (Kasinathand Kanwar, 1993). It cannot be excluded that nephrin is a proteoglycan,as it has several SG consensus sites for heparan sultate side chains,including the triplet SGD which is the major attachment sequence for thethree large heparan sulfate side chains in the basement membraneproteoglycan perlecan (Noonan et al., 1991; Kallunki and Tryggvason,1992; Dolan et al., 1997). However, thus far no Ig-like receptors havebeen reported to contain glycosaminoglycans.

How does nephrin function and what is its role in glomerular function? Avast majority of similar receptors interact with other membrane proteinsin a homo- or heterophilic manner. However, some of the receptors havebeen shown to interact with extracellular matrix (ECM) proteins. Forexample, the myelin-associated glycoprotein MAG whose extracellulardomain contains five Ig-like domains, interacts with different types ofcollagens and glycosaminoglycans (Fahrig et al., 1987). Furthermore, theaxonal glycoprotein F11 and the deleted in colorectal cancer (DCC)protein have both been shown to bind tenascins and netrins, respectively(Zisch et al., 1992; Pesheva et al., 1993; Keino-Masu, 1996). Since itis possible that nephrin either binds another membrane protein or aprotein of the ECM, which in this case would be the GBM, it will beimportant to localize nephrin by immunoelectron microscopy beforeembarking on the search for a specific ligand.

Whatever its function, the in situ hybridization analyses stronglysuggested that nephrin is produced in glomerular epithelial cells thatform the foot processes partially covering the outside of the glomerularcapillaries. The ultimate filtration barrier for plasma macromoleculesis located in the diaphragm covering the slit pores between the footprocesses. In NPHS1 and nephrotic syndromes of other causes, fusion ofthe foot processes is a general finding, and the structure or functionof the slit pores are somehow affected with proteinuria as a result. Itis proposed that the plasma membrane protein nephrin is important formaintaining the integrity of the foot processes of glomerular epithelialcells, or is crucial for their anchorage to components of the GBM.

Conclusions

The identification of the NPHS1 gene will immediately find applicationsfor diagnosis of the disease. Studies on the gene product nephrin, aputative cell adhesion and signaling receptor, may also provide a key tonew fundamental knowledge on the molecular mechanisms of glomerularfiltration, which despite decades of research are still poorlyunderstood. As abnormal function of the filtration barrier is a majorcomplication in many clinically important kidney diseases, such asdiabetic nephropathy, nephrotic syndromes and glomerulonephritides, thepresent work is likely to have a more general impact on clinicalnephrology. Immediate questions relate to the developmental expressionand location of the protein, which would require the generation ofantibodies and nucleotide probes for studies in animal and cell culturesystems.

EXAMPLE 6

Genetic Screening for Basement Membrane Disease

With the identification and characterisation of nephrin as a criticalcomponent in basement membrane disease associated with glomerularnephropathy, it is now possible to screen individuals, both pre- andpost-natal screening, for susceptibility for basement membrane diseaseby detecting mutated nephrin gene or protein. Such information will beuseful to medical practitioners for the future diagnosis of diseaseconditions in screened individuals, and for planning preventativemeasures for the possible containment of future disease. Suchinformation will be useful for the diagnosis of currently active diseaseconditions. The present invention allows for the diagnosis of currentlyactive disease conditions, as being related to basement membrane diseaseby detecting mutated nephrin gene or protein. The discovery of thenephrin gene provides a means for detecting the presence of the nephringene in individuals, and for the determination of the presence of anymutations in said gene. Such means for detection comprises nucleic acidshaving the entire nephrin gene sequence, or fragments thereof which willspecifically hybridize to said nephrin gene, or mRNA transcripts fromsaid nephrin gene under stringent conditions. An additional means fordetection of the nephrin gene and mutations therein comprise specificcontiguous fragments of said gene, and complementary gene sequence,which can be combined for use as primers for amplifying the targetedgene sequence. Said means for detection of mutations in a nephrin genealso comprise direct hybridization of normal gene with target gene andsubsequent detection of successful hybridization. In all cases, thetarget gene may be amplified or unamplified DNA or RNA isolated from theindividual to be tested.

Antibody Screening of Tissues and Samples

By having the NPHS1 gene sequence, it is well within the skill of one inthe art to use existing molecular biology and biochemical techniques toconstruct and use an expression vector which will produce recombinantnephrin protein, or fusion protein, purify this protein, and produceantibodies specifically reactive with nephrin. The expression ofproteins in bacterial, yeast, insect and mammalian cells is known in theart. It is known in the art how to construct and use expression vectorsin which the expressed gene contains one or more introns. The productionof monoclonal antibodies is well known in the art, and the use ofpolyclonal and monoclonal antibodies for immunohistochemical detectionof protein in tissue samples is a routine practice. A wide variety ofdetectable labels are available for use in immunohistochemical stainingand immunoassays for detection of protein in samples such as homogenisedtissue, blood, serum, urine or other bodily fluids.

One of ordinary skill in the art will be able to readily use theteachings of the present invention to design suitable assays anddetection schemes for practising the screening methods contemplated bythe present invention.

Gene Therapy

Given the teaching of the present invention, it will be possible toaddress deficiencies in Nephrin gene or protein by gene therapy ortherapy using recombinant protein. Methods for the administration ofprotein and gene therapy are known in the art.

GenBank Accession Numbers

The accession numbers for the cosmid clones characterised are:

-   F19541=U95090, R33502=AC002133, R28051=AD000864, F19399=AD000833,    R31158=AD000827, R31874=AD000823. The accession for the nephrin cDNA    sequence is AF035835.

One of ordinary skill in the art will be able to readily use theteachings of the present invention to design and construct suitablenucleic acid sequences which will be the functional equivalents of thosedisclosed. One of ordinary skill in the art will know that there existsmany allelic variants of the disclosed nucleic acid sequences whichstill encode for a nephrin protein with equivalent function. Theteaching of the present invention allows for the discovery of mutationsin the nephrin gene and the modified protein therein encoded.

EXAMPLE 7

Screening for Small Molecule Therapeutics

With the identification and characterisation of nephrin as a criticalcomponent in kidney pathology and proteinuria, and thus implicated inmany kidney diseases, it is now possible to screen for small moleculetherapeutics using nephrin and the nephrin gene. Screening for suchtherapeutics can be accomplished by sequential selective screening foractivity and molecules which specifically hybridize to nephrin, or whichspecifically effect the expression of the nephrin gene. Selectivescreening can be performed on pools of small molecule compoundsgenerated by standard combinatorial chemistry, on known molecules, or incombination with computer modeling of the nephrin protein structure andrational drug design. Such methods and techniques are known in the art.

Literature Cited

-   Altschul, S. F., Gish, W., Miller, W., Myers, E. W., and    Lipman, D. J. (1990) Basic local alignment search tool. J. Mol.    Biol. 215, 403-10-   Appel, R. D., Bairoch, A. and Hochstrasser, D. F. (1994) A new    generation of information retrieval tools for biologists: the    example of the ExPASy WWW server. Trends Biochem. Sci. 19: 258-260-   Barker, D., Hostikka, S. L., Zhou, J., Chow, L. T., Oliphant, A. R.,    Gerken, S. C., Gregory, M. C., Skolnick, M. H., Atkin, C. L. and    Tryggvason, K. (1990) Identification of mutations in the COL4A5    collagen gene in Alport syndrome. Science 248, 1224-1227-   Brummendott, T., and Rathjen, F. G. (1994) Cell adhesion molecules    1: Immunoglobulin superfamily. Protein profile 1, 951-1058.-   Burge, C. and Karlin, S. (1997) Prediction of complete gene    structures in human genomic DNA. J. Mol. Biol. 268, 78-94.-   de la Chapelle, A. (1993) Disease gene mapping in isolated human    populations: the example of Finland. J. Med. Genet. 30:857-865-   Dolan, M., Horchar, T., Rigatti, B., and Hassell, J. R. (1997)    Identification of sites in domain I perlecan that regulate heparan    sulfate synthesis. J. Biol. Chem. 272, 4316-4322-   Fahrig, T., Landa, C., Pesheva, P., Kühn, K., and    Schacher, M. (1987) Characterization of binding properties of the    myelin-associated glycoprotein to extracellular matrix constituents.    EMBO J. 6, 2875-2883.-   Fuchshuber, A., Niaudet, P., Gribouval, O., Genevieve, J., Gubler,    M-C., Broyer, M. and Antignac, C. (1996) Congenital nephrotic    syndrome of the Finnish type: linkage to the locus in a non-Finnish    population. Pediatr. Nephrol. 10: 135-138-   Genetics Computer Group, Program manual for the Wisconsin Package,    Version 9, December 1996, 575 Science Drive, Madison, Wis., USA    53711-   Hästbacka, J., de la Chapelle, A., Mahtani, M. M., Clines, G.,    Reeve-Daly, M. P., Daly, M., Hamilton, B. A., Kusumi, K., Trivedi,    B., Weaver, A., Coloma, A., Lovett, M., Buckler, A., Kaitila, I.,    and Lander, E. S. (1994) The diastrophic dysplasia gene encodes a    novel sulfate transporter: positional cloning by fine-structure    linkage disequilibrium mapping. Cell 78, 1073-1087-   Holmberg, C., Antikainen, M., Ronnholm, K., Ala-Houhala, M. and    Jalanko, H. (1995) Management of congenital nephrotic syndrome of    the Finnish type. Pediatr. Nephrol. 9: 87-93-   Huttunen, N. P., Rapola, J., Vilska, J. and Hallman, N. (1980) Renal    pathology in congenital nephrotic syndrome of the Finnish type: a    quantitative light microscopic study on 50 patients. Int. J.    Pediatr. Nephr. 1: 10-16-   Ikonen, E., Baumann, M., Grön, K., Syvänen, A-C., Enomaa, N.,    Halila, R., Aula, P. and Peltonen, L. (1991) Aspartylglucosaminuria:    cDNA encoding human aspartylglucosaminidase and the missense    mutation causing the disease. EMBO J. 10: 51-58-   Jurka, J., Klonowski, P., Dagman, V., Pelton, P. (1996) CENSOR—a    program for identification and elimination of repetitive elements    from DNA sequences. Computers and Chemistry Vol. 20 (No. 1):    119-122.-   Kallunki, P., and Tryggvason, K. (1992) Human basement membrane    heparan sulfate proteoglycan core protein: a 467 kD protein    containing multiple domains resembling elements of the low density    lipoprotein receptor, laminin, neural cell adhesion molecules, and    epidermal growth factor. J. Cell Biol. 116, 559-571-   Kasinath, B. S. and Kanwar, Y. S. (1993) Glomerular basement    membrane: biology and physiology. In: Molecular and cellular aspects    of basement membranes (D. Rorhbach and R. Timpl, eds), Academic    Press, pp. 89-106.-   Kawai, S., Nomura, S., Harano, T., Fukushima, T., & Osawa, G. (1996)    The COL4A5 gene in Japanese Alport syndrome patients: spectrum of    mutations of all exons. Kidney Int. 49, 814-822-   Keino-Masu, K., Masu, M. Hinck, L., Leonardo, E. D., Chan, S. S. Y.,    Culotti, J. G., and Tessier-Lavigne, M. (1996) Cell 87, 175-185-   Kestilä, M., Männikkö, M., Holmberg, C., Korpela, K., Savolainen, E.    R., Peltonen, L. and Tryggvason, K. (1 994a) Exclusion of eight    genes as mutated loci in congenital nephrotic syndrome of the    Finnish type. Kidney Int. 45, 986-990-   Kestilä, M., Männikkö, M., Holmberg, C., Gyapay, G., Weissenbcah,    J., Savolainen, E. R., Peltonen, L. and Tryggvason, K. (1994b)    Congenital nephrotic syndrome of the Finnish type maps to the long    arm of chromosome 19. Am. J. Hum. Genet. 54, 757-764-   Knebelmann, B., Breillat, C., Forestier, L., Arrondel, C.,    Jacassier, D., et al. (1996) Spectrum of mutations in the COL4A5    collagen gene in X-linked Alport syndrome. Am. J., Hum. Genet. 59,    1221-1232-   Koskimies, 0. (1990) Genetics of congenital and early infantile    nephrotic syndromes. In: Spitzer, A., Avner, E. D. (eds) Inheritance    of kidney and uritary tract diseases. Kluwer, Boston, Dordrecht and    London, p. 131-138-   Laine, J., Jalanko, H., Holthofer, H., Krogerus, L., Rapola, J., von    Willebrand, E., Lautenschlager, I., Salmela, K. and    Holmberg, C. (1993) Post-transplantation nephrosis in congenital    nephrotic syndrome of the Finnish type. Kidney Int. 44: 867-874-   Lenkkeri, U., Kestilä, M., Lamerdin, J., McCready, P., Adamson, A.,    Olsen, A. and Tryggvason, K. Structure of the human amyloid    precursor like protein gene APLP1 at 19q13.1. Human Genetics, in    press-   Ljungberg, P., Jalanko, H., Holmberg, C. and Holthofer, H. (1993)    Congenital nephrosis of the Finnish type (CNF): matrix components of    the glomerular basement membranes and of cultured mesangial cells.    Histochem. J. 25: 606-612-   Mahan, J. D., Mauer, S. M. Sibley, R. K. and Vernier, R. L. (1984)    Congenital nephrotic syndrome: Evolution of medical management and    results of renal transplantation. J. Pediatr. 105: 549-557-   Männikkö, M., Kestilä, M., Holmberg, C., Norio, R., Ryynnen, M.,    Olsen, A., Peltonen, L. and Tryggvason, K. (1995) Fine mapping and    haplotype analysis of the locus for congenital nephrotic syndrome on    chromosome 19q13.1. Am. J. Hum. Genet. 57: 1377-1383-   Männikkö, M., Kestilä, M., Lenkkeri, U., Alakurtti, H., Holmberg,    C., Leisti, J., Salonen, R., Aula, P., Mustonen, A., Peltonen, L.    and Tryggvason, K. (1997) Improved prenatal diagnosis of the    congenital nephrotic syndrome of the Finnish type based on DNA    analysis, Kidney Int. 51: 868-872-   Martin, P., Heiskari, N., Hertz, J.-M-, Atkin, C., Barker, D., et    al. Survey of mutations in the COL4A5 collagen gene in patients with    suspected Alport syndrome: PCR amplification and direct sequencing    of all 51 exon regions reveals over 80 mutation detection rate.    (manuscript).-   Noonan, D. M., Fulle, A., Valente, P., Cai, S., Horigan, E., Sasaki,    M., Yamada, Y., and Hassell, J. R. (1991) The complete sequence of    perlecan, a basement membrane heparan sulfate proteoglycan, reveals    extensive similarity with laminin A chain, low density lipoprotein    receptor, and the neural cell adhesion molecule. J. Biol. Chem. 266,    22939-22947-   Norio, R. (1966) Heredity on the congenital nephrotic syndrome. Ann.    Paediatr. Fenn. 12 (suppl 27):1-94-   Olsen, A., Georgescu, A., Johnson, S. and Carrano, A. V. (1996)    Assembly of a 1-Mb restriction-mapped cosmid contig spanning the    candidate region for Finnish congenital nephrosis (NPHS1) in    19q13.1. Genomics 34:223-225-   Pesheva, P., Gennarini, G., Goridis, C., and Schacher, M. (1993) The    F3/11 cell adhesion molecule mediates the repulsion of neurons by    the extracellular matrix glycoprotein J1-160/180. Neuron 10, 69-82-   Pekkarinen P., Kestilä M., Hakola P., Järvi O., Tryggvason, K. and    Palotie L. Fine-scale mapping of the PLO-SL locus. (manuscript)-   Rapola, J., Huttunen, N. P. and Hallman, N. (1992) Congenital and    infantile nephrotic syndrome. In: Edelman C M (ed.) Pediatric Kidney    Disease. 2nd ed. Little, Brown and Company, Boston. Vol 2: 1291-1305-   Renieri, A., Bruttini, M., Galli, L., Zanelli, P., Neri, T., et    al. (1996) X-linked Alport syndrome: an SSCP-based mutation survey    over all 51 exons of the COL4A5 gene. Am. J. Hum. Genet. 58,    1192-1204-   Solovyev, V. V., Salamov, A. A., Lawrence, C. B. (1994) Predicting    internal exons by oligonucleotide composition and discriminate    analysis of spliceable open reading frames. Nucl. Acids Res. 22(24):    5156-5163-   Tryggvason, K. (1996) Mutations in type IV collagen genes in Alport    syndrome. In: Molecular pathology and Genetics of Alport syndrome    (ed. K. Tryggvason). Contrib. Nephrol., 117, 154-171, Karger, Basel-   Tryggvason, K. and Kouvalainen, K. (1975) Number of nephrons in    normal human kidneys and kidneys of patients with the congenital    nephrotic syndrome. Nephron 15: 62-68-   Uberbacher, E. C. and Mural, R. J. (1991) Locating protein-coding    regions in human DNA sequences by a multiple sensor-neural network    approach, Proc. Natl Acad. Sci. USA 88: 11261-11265-   Zisch, A. H., D'Allessandri, L., Ranscht, B., Falchetto, R.,    Winterhalter, K. H., and Vaughan, L. (1992) Neuronal cell adhesion    molecule contactin/F11 binds to tenascin via its immunoglobulin-like    domains. J. Cell Biol. 119, 203-213

1. A method for detecting the susceptibility to, or the presence ofexisting basement membrane disease in an individual comprising the stepsof providing a nucleic acid sample obtained from the individual, whereinthe sample contains the NPHS1 gene, or fragments thereof, and detectinga mutation in said gene, or some fragment thereof, wherein if anindividual is susceptible to, or has the basement membrane disease, theindividual has at least one mutation in said gene.
 2. The method ofclaim 1, wherein said basement membrane disease is congenital nephroticsyndrome of the Finnish type.
 3. The method of claim 1, wherein theNPHS1 gene comprises a nucleic acid sequence that encodes nephrinprotein comprising SEQ ID NO:
 2. 4. The NPHS1 gene of claim 1, whereinthe nucleic acid sequence comprises SEQ ID NO:
 1. 5. The method of claim1, wherein the detection of a mutation in the NPHS1 gene comprisesanalyzing a test nucleic acid sample obtained from the individual for atleast one mutation of the NPHS1 gene, or a fragment thereof, andcomparing the results of the analysis of the test sample with theresults of the analysis of a control sample, wherein the control samplecomprises a NPHS1 gene comprising SEQ ID NO: 1 without a mutation. 6.The method of claim 5 wherein the analysis comprises determining thenucleic acid sequence of the nucleic acid in the test and controlsamples.
 7. The method of claim 6, wherein the NPHS1 gene is amplifiedprior to analysis.
 8. A method for determining that an individual is notat risk for developing congenital nephrotic syndrome of the Finnishtype, comprising analyzing a nucleic acid sample obtained from theindividual wherein the sample contains the NPHS1 gene, or fragmentsthereof, to detect a mutation in said gene, or some fragment thereof,wherein the individual is not at risk for developing the syndrome if theNPHS1 gene or fragment does not have a mutation.
 9. The method of claim8, wherein the NPHS1 gene comprises a nucleic acid sequence that encodesnephrin protein comprising SEQ ID NO:
 2. 10. The method of claim 8,wherein the NPHS1 gene comprises SEQ ID NO:
 1. 11. A method ofdetermining whether an individual is at risk for developing a congenitalnephrotic syndrome of the Finnish type, comprising analyzing a nucleicacid sample obtained from the individual, wherein the sample containsthe NPHS1 gene, or fragments thereof, to detect a mutation in said gene,or fragment thereof, wherein the individual at risk for developing thesyndrome has at least one mutation in the NPHS1 gene or fragmentthereof.
 12. The method of claim 11, wherein the NPHS1 gene comprises anucleic acid sequence that encodes nephrin protein comprising SEQ ID NO:2.
 13. The method of claim 11, wherein the NPHS1 gene comprises SEQ IDNO:
 1. 14. The method of claim 13, wherein the NPHS1 gene is amplifiedprior to analysis.
 15. A method for detecting the presence or absence ofa mutation in a NPHS1 gene, comprising the steps of: a) analyzing anucleic acid test sample containing the NPHS1 gene or fragments thereof,for at least one mutation of the gene or fragment thereof; b) comparingthe results of the analysis of the test sample of step a) with theresults of the analysis of a control sample, wherein the control samplecomprises a NPHS1 gene or fragment thereof without a mutation; and c)determining the presence or absence of at least one mutation in theNPHS1 gene or fragment thereof in the test sample.
 16. The method ofclaim 15 wherein the NPHS1 gene comprises a nucleic acid sequence thatencodes nephrin protein comprising SEQ ID NO:
 2. 17. The method of claim15, wherein the NPHS1 gene comprises SEQ ID NO:
 1. 18. The method ofclaim 15, wherein the NPHS1 gene is amplified prior to analysis.